Fig. 5: Changes in cell resorption observed with exposure to simulated microgravity.
a To simulate a reduction in the force of gravity that the femoral head trabeculae are normally adapted to resist, particularly when upright (left), organoids were placed either in static, typical culture conditions, where they experience the normal gravitational pull, or in dynamic, rotational conditions, inside a NASA-synthecon reactor vessel, where a laminar movement of the culture medium suspends constructs in a state of orbital buoyancy (right). During this culture model, the forces acting on the trabecular organoids, resulting from the gravitational force (Fg), centrifugal forces (Fc) and hydrodynamic drag forces (Fd) (b) balance each other during each orbital revolution (c), thus preventing sedimentation and simulating weightlessness. Following 6 days of culture under these conditions, resorption induced by cells can be observed in osteoclastic and osteoblastic-osteoclastic constructs (d, red arrows and box), which appears longitudinally extended in morphology (e). This resorption pattern resembles a second class of lacunae encountered in trabecular bone from human femoral heads (f) and is different structurally from the type encountered in constructs cultured under static conditions (g, h). Extracts from the incubation medium of constructs subjected to simulated microgravity were taken as pellets (i), or immuno-purified from liquid phase (j). In both cases, large protein complexes were detected between 60 and 70 kDa, containing an association of the target protein, native cell and medium proteins (j right, black rectangles). These regions were probed with antibodies during western blotting (i), or the protein was immuno-selected using magnetic isolation (j, left). The protein Sclerostin (i) was detected in osteoblastic samples, but additionally in osteoclast and mixed samples/medium. This pattern was also observed in concentrated medium samples (j, left—immuno-selected samples and right—all samples including medium). RANKL (i) was not expressed in osteoblast constructs, in the absence of osteoclasts. RANKL was also detected in concentrated medium samples, in all but osteoblast samples (j). The endocrine protein PTHR1 and integrin CX43 (i) were only detected in osteoclastic constructs. Figure f is taken from Gentzsch et al.75 by permission from Springer Nature. Scale bars as indicated.
